We firstly checked the most prevalent (appeared in more than 50 populations) and abundant variants (average allele frequency > 0.2) variants.
For gene peg.962 (RND efflux system inner membrane transporter), multiple variants (N = 9) were detected in six different positions in ALE2.0, with most of them (seven out of nine) dominate the populations (with allele frequency higher than 0.9).
The first gene shown as peg.1114, turns out to be a non-coding region variants at position 1,425,628 located in the upstream (d = 40 bp) of gene peg.1113, which is identified as Lipoprotein in UniProt, which is confirmed by the String as it neighbored coding gene of Fructokinase (peg. 1114). In B. uniformis genome, in total, two copies of Fructokinase gene were annotated (the other is peg. 2097).
For mutations around gene peg.1246 and peg.1250, as we could see, most of them are within the coding region, with only 2 in non-coding region. For gene peg.1246 (Fic family protein), most of the variants have AF < 0.1, with one frame shift variants (at the position 1,600,695) above slightly. For the second gene (peg.1250), no function is annotated and blasting again UniProt shows alignment to DUF4468 domain-containing protein. A frame shift variant with AF = 1 is detected in multiple samples, however, no replicate were found for any compound.
Variants before and within the Glutamyl-tRNA synthetase gene (peg.1351) show a low allele frequency in general. Only two missense variants were detected within the coding region (in Montelukast and Acarbose) at very close positions in the end of the protein sequnce (501/502 out of 508 amino acids).
For mutations around gene peg.1416 and peg.1417, both genes are on the + strand, which means, only one synonymous mutation was detected in the end of gene peg.1416 (Histidine ammonia-lyase (EC 4.3.1.3);Ontology_term=KEGG_ENZYME:4.3.1.3). For gene peg.1417, which is annotated as transcriptional regulator AcrR family, which is reported to be a regulation system that confers resistance to the antibiotic tetracycline Kang et al., 2019 and regulate the AcrAB-TolC efflux pump (peg. 1418, 1419, and 1420). Notably, variants before and within gene AcrR are mainly detected in Xanthan gum evolved samples. All populations (N = 8 replicates with two different concentrations of Xanthan gum) have either non coding region or frame shift variant detected.
Variants before the gene (peg.1537) were detected at four different positions (1914983, 1914985, 1914986, and 1914999), among those, variants at three positions were also detected in parental populations, therefore, we assume these variants might not affect the growth or resistance of the population.
For mutations within gene peg.1554 (Integrase) and peg.1555 (Outer membrane TonB-dependent transporter utilization system for glycans and polysaccharides/PUL SusC family), variants are concentrated in several positions. Frame shift variant was detected at the position 1,931,428 within the integrase gene, with the same variant detected in 1B9 (one of the parental population).
For TonB gene, only synonymous and missense variants were detected.
Variants were detected before gene peg.2424 (unknown, 68 amino acids), with AF > 0.1 found in higher concentration PFOA (ALE 2.0) and Xanthan gum.
Variants were detected before gene peg.2727 (ompA, outer membrane protein, strand -), with AF > 0.1 found in many populations including parental and control samples.
Variants were detected around these three genes, with a variant detected in PFNA (ALE 2.0) before gene peg.2981 (UDP-glucose 4-epimerase (EC 5.1.3.2);Ontology_term=KEGG_ENZYME:5.1.3.2) at 3,543,736.
For gene peg.3135 (3683196 to 3683321, + strand), pipetide with 42 amino acids, all variants were insertion and detected at the position 3,683,056, 140 bp upstream of peg.3135 and 82 bp downstream of previous gene (peg.3134, Methyl-directed repair DNA adenine methylase (EC 2.1.1.72);Ontology_term=KEGG_ENZYME:2.1.1.72).
Variants were detected before and within this gene, annotated as Acyl_transf_3 from Acyltransferase family (3929637 to 3929819, + strand).